Cardiovascular disease (CVD) is the leading cause of death in the world. Many risk factors have been identified as well as foods thought to be associated with increased CVD risks such as meats, high-fat dairy products, saturated fats and egg yolks. We described a previously unrecognized A meta-organismal pathway involving gut microbial (gut flora) dependent metabolism of trimethylamine-containing nutrients abundant in these foods (choline and l- carnitine) to trimethylamine (TMA) that is converted by the host liver enzymes to trimethylamine-N-oxide (TMAO) which is an atherogenic compound. We have shown using basic, animal model and human clinical studies that gut flora take part in development of CVD and its associated unfavorable events. Microbial enzymes responsible for generating TMA from various dietary TMA-containing nutrients are TMA lyases. Two microbial TMA lyases have been identified and cloned, CutC/D and CntA/B, which show strict substrate specificity for cleaving choline and carnitine, respectively. We recently cloned and characterized a related TMA lyase, YeaW/X, which demonstrates broad substrate specificity. In unpublished studies we see additional TMA lyase substrate activities in gut microbiota based on substrate preferences that are not catalyzed by either CutC/D or CntA/B. We propose to examine the relationship between specific microbial TMA lyases and the increased blood TMAO levels and development of atherosclerosis. We will further define the microbial TMA lyases responsible for TMA and TMAO generation in vivo, and the involvement of specific microbial TMA complexes (YeaW/X, CutC/D, and CntA/B) in diet-induced atherosclerosis using animal models. We will also test the potential of microbial choline TMA lyase complex pharmacologic manipulation in impacting host diet-induced impairment in reverse cholesterol transport and atherosclerosis. The proposed studies will help define the contribution that individual TMA lyase enzymes in vascular disease, and provide important nutritional and pharmacological insights into the prevention and treatment of atherosclerosis.